Detector Arrangement for Blood Culture Bottles With Colorimetric Sensors

ABSTRACT

A detector arrangement for a blood culture bottle incorporating a colorimetric sensor which is subject to change of color due to change in pH or CO 2  of a sample medium within the blood culture bottle. The detector arrangement includes a sensor LED illuminating the colorimetric sensor, a reference LED illuminating the colorimetric sensor, a control circuit for selectively and alternately activating the sensor LED and the reference LED, and a photodetector. The photodetector measures reflectance from the colorimetric sensor during the selective and alternating illumination of the colorimetric sensor with the sensor LED and the reference LED and generates intensity signals. The reference LED is selected to have a peak wavelength of illumination such that the intensity signals of the photodetector from illumination by the reference LED are not substantially affected by changes in the color of the colorimetric sensor.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/400,001, entitled “Detector Arrangement for BloodCulture Bottles With Colorimetric Sensors”, filed Jul. 20, 2010, whichis incorporated herein.

BACKGROUND

Bottles for culturing of blood for the presence of microorganism andrelated instruments for analyzing such bottles in a noninvasive mannerare known in the art and described in the patent literature. See U.S.Pat. Nos. 5,858,769; 5,795,773; 4,945,060; 5,094,955; 5,164,796;5,217,876; and 5,856,175. The bottles and instruments of theabove-listed patents have been commercialized with success by thepresent assignee under the trademark BacT/ALERT.

The bottles described in these blood culture instruments utilizecolorimetric sensors placed in the bottom of the bottle and in contactwith the sample media to determine the presence/absence of bacterialgrowth. Once a clinical/industry sample is added to the liquid growthmedia present in the bottle and incubation occurs, the concentration ofcarbon dioxide increases as the number of microorganisms increase;carbon dioxide is a respiration by-product of bacterial growth.Alternatively, changes to the media pH that are related to the growth ofmicroorganisms can also be monitored by the sensor. The basic operationof the BacT/ALERT sensor and monitoring electronics is described in U.S.Pat. No. 4,945,060 and also in an article by Thorpe et. al. in“BacT/Alert: an Automated Colorimetric Microbial Detection System” whichwas published in the Journal of Clinical Microbiology, July 1990, pp.1608-12. The '060 patent and the Thorpe et al. article are incorporatedby reference here.

The basic colorimetric sensing system described in the '060 patent isshown in FIG. 1 of the appended figures. A red Light Emitting Diode(LED) (4) shines onto the bottom of the BacT bottle (1). A colorimetricsensor (2) is deposited onto the bottom of the bottle (1). The LED lightimpinges on the sensor at a 45 degree angle relative to the bottomsurface of the bottle (1). The majority of the light penetrates thestructure of the bottle and impinges on the colorimetric sensor (2).Part of the light will reflect off the plastic bottle material andsensor (2) at 45 degrees to the bottom surface of the bottle, but in anopposite direction to the impinging light (e.g. the angle of reflectionis equivalent to the angle of incidence). Much of the remaining light isscattered from the surface and interior of the sensor. The sensor (2)changes its color as the percentage of CO₂ in the bottle varies from 0%to 100%; the color varies from blue to yellow, respectively. A siliconphotodetector (5) “stares” (i.e., continuously monitors the scatteredintensity signal) at the region in the sensor (2) where the light fromthe LED interacts with the sensor. The intensity of the scattered lightthat is detected by the photodetector is proportional to the CO₂ levelwithin the bottle (1). FIG. 1 also shows the associated electronicsincluding a current source (6), current-to-voltage converter (7) and lowpass filter (8).

FIG. 2 is a plot of the signal received by the photodetector (5) ofFIG. 1. The data was collected using a fiber optic probe in place of thephotodetector (5) in FIG. 1. The fiber optic probe is routed to avisible light spectrometer, which shows the scattered light as afunction of intensity (Reflectance Units) and wavelength. The shape ofeach curve is the convolution of the LED intensity distribution with thereflectivity of the colorimetric sensor (2) at a specified CO₂ level.

When the silicon photodetector (5) of FIG. 1 is substituted for thefiber optic probe, a photocurrent is generated by the photodetector thatis proportional to the integrated wavelength signal shown in FIG. 2. Inother words, the silicon photodetector integrates the spectral responseinto a photocurrent. In turn, this photocurrent is converted into avoltage signal using a transimpedance amplifier.

While the BacT/ALERT sensing system of FIG. 1 is robust and has beenused in blood culture systems successfully for many years, it does havea few areas for improvement. First, if the blood culture bottle (1)moves in the cell (e.g. displacement in the z-axis so that it shiftsaway from the position of the photodetector), the system (as it iscurrently implemented) detects this movement as a reduction inintensity. However, this reduction in intensity is interpreted by theinstrument as reduction in CO₂ level in the bottle, which may not infact be occurring. Since this effect is counter to the effect of abottle's reflectivity increasing as carbon dioxide content increases(signifying bacterial growth), it is possible that the system wouldtreat a translating bottle as having no growth (i.e., a false negativecondition).

Likewise, as the instrument ages in the clinical laboratory, the opticalsystem may collect dust or optical materials experience reducedtransmissivity as a function of time. For example, as plastics age,their transmissivity can be reduced by the effects of light, particulatebuildup (dust) or repeated use of cleaning agents. These effects wouldnot affect readings but would manifest as a drift in the response of thesystem. Periodic calibration checks could compensate for this drift.Thus, there is a long-felt but unmet need to have a real-time monitor ofthe transmission in the optical system and the capability to adjust orcompensate for some of these sources of error, particularly thesituation where the bottle is not fully installed in the receptacle andis not at the nominal or home position (has some Z-axis displacementaway from the optical detector arrangement).

Other prior art of interest includes the following U.S. Pat. Nos.7,193,717; 5,482,842; 5,480,804; 5,064,282; 5,013,155; 6,096,2726;6,665,061; 4,248,536 and published PCT application WO 94/26874 publishedNov. 24, 1994.

SUMMARY

An improved detection arrangement for blood culture bottle incorporatingcolorimetric sensors is disclosed.

The detection arrangement includes photodetector, a sensor LED and areference LED, and a control circuit for selectively and alternatelyactivating the sensor LED and the reference LED to illuminate thecolorimetric sensor. The sensor LED functions like the LED of FIG. 1 andis used to determine the change in the colorimetric sensor color. Thephotodetector monitors the reflectance from the sensor when illuminatedby the sensor LED by monitoring intensity changes. The reference LED isselected to have a wavelength such that the intensity readings of thephotodetector from illumination by the reference LED are not affected bychanges in the color of the colorimetric sensor. As such, the referenceLED can be used as a reference, with the photodetector readings duringillumination by the reference LED unaffected by changes in CO₂concentration within the bottle. It has been found that wavelengths inthe near infra-red (peak λ for the LED between 750 and 950 nm) aresuitable for the reference LED.

The reference LED is useful to indicate if the distance between thebottle and the detector subassembly changes, ambient lighting conditionschange, or anything within the physical optical path between the sensorLED, the bottle and the photodetector changes. Since a change in thereference LED is not dependent on the state of the colorimetric sensor,the reference LED can provide information about changes in the opticalsystem that are not related to microorganism growth so that suchnon-growth related changes from the system can be discriminated fromgrowth-related changes. This feature helps reduce the false-positiverate in the system and improves sensing accuracy and reliability.

In use, the sensor LED and reference LED are illuminated alternately andrepeatedly, e.g., in a time division multiplexed manner. Thephotodetector signals from such sequential illuminations are fed to acomputer. The computer monitors changes in the photodetector signal whenthe reference LED illuminated; these changes would indicate a change inthe bottle position or the optical system. The computer can compensatethe sensor LED signals according to derived calibration relationshipsbetween the sensor LED and reference LED signals, e.g., due to offset ofthe bottle position in the detection system from a home or nominalposition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a known sensor and detector arrangement forblood collection bottles as described in U.S. Pat. No. 4,945,060.

FIG. 2 is plot of reflectance of a colorimetric sensor on a spectrometerin place of the photodetector of FIG. 1 as a function of wavelength andCO₂ concentration.

FIG. 3 is a sensor and detector arrangement for blood collection bottlesin accordance with the present disclosure.

FIG. 4 is a plot of intensity signals from the photodetector of FIG. 3for sensor LED and reference LED illumination of the colorimetric sensorover 0-100% CO₂ range present within the bottle.

FIG. 5 is a graph of photodetector intensity signals for the sensor LEDand reference LED as a function of bottle displacement from nominal orhome position in which the bottle is in its designed position proximateto the detection system of FIG. 3.

FIG. 6 is a plot of photodetector intensity signals for the sensor LEDand reference LED as a function of time during conditions of microbialgrowth with the bottle.

FIG. 7 is a block diagram of the electronics operating the sensorarrangement of FIG. 3.

FIG. 8 is a graph of the duty cycle of the reference and sensor LED ofFIG. 3, showing the time division multiplexing method of operation. Thewidth of the pulses representing the duty cycle is not to scale; in onepossible embodiment the duty factor is 33 percent: ⅓ of the time thereference LED is illuminated, ⅓ of the time the sensor LED isilluminated, and ⅓ of the time neither LED is illuminated to enable a“dark” measurement to be made.

DETAILED DESCRIPTION

The invention involves the use of secondary LED as a light source tocompensate for non-Liquid Emulsion Sensor (LES) changes to the opticalsystem. A block diagram of the optical configuration is shown in FIG. 3.The configuration is for testing a bottle 1 having a colorimetric LES 2incorporated within the bottle 1. The configuration includes a sensorLED 4, an IR reference LED 10, and a photodetector 5 generatingintensity signals. Both LEDs 4 and 10 are angled at 45 degrees inrelation to the bottom surface of the bottle as shown in FIG. 3. Thereflectivity of the bottle bottom and LES 2 is measured sequentially, bymeans of a control circuit (42, FIG. 7) which selectively andalternately activates the sensor LED and the reference LED. For example,the sensing or red LED 4 is turned on and the reflected signal ismeasured by the photodetector 5. The sensing LED 4 is then extinguished.The reference LED 10 is then illuminated and the same photodetector 5measures the reflected light. Then it is extinguished, and the processis repeated. This approach is also referred to as a time-divisionmultiplexed scheme, which is shown in FIG. 8 and will be described infurther detail below.

As noted above, the LEDs 4 and 10 are oriented at a 45 degree anglerelative to the bottom of the bottle. This is so that the reflection offof the bottom surface of the bottle is not strongly coupled into thephotodetector 5. The angle of incidence=angle of reflection so thatlight striking the bottle bottom will exit off at 45 degrees and willnot strongly affect the photodetector reading (since scattered lightfrom the LES is only of interest). The LEDs have a spatial emissionangle of 15-17 degrees; i.e., the LEDs emit light in a cone that isdefined by Peak Emission and Full-Width angle at half maximum power; theangle of the cone is in the range of 15-24 degrees.

Testing was performed on a variety of LED colors, and it was found thatnear-infrared LEDs (peak wavelength from 750-950 nm) reflectivity weremarginally effected by the LES color changes. All other wavelengths oflight had a negative or positive change in reflectivity as the CO2 levelwas changed from 0% to 100%. This effect minimizes at wavelengths beyondabout 750 nm (near-infrared LED) as is shown in Table 1.

TABLE 1 Photodetector output (volts) with CO2 spiked bottles For sensing(RED) LED and reference (IR) LED CO₂ Sensing LED Reference LED LevelSamples Mean Std. Dev. Mean Std. Dev. 0% 390 0.65838 0.00045 2.325390.00045 2% 390 0.84627 0.00048 2.25763 0.00048 15% 390 1.29105 0.000472.40419 0.00048 100% 390 1.92822 0.00063 2.29345 0.00050

FIG. 4 shows the graphical equivalent of Table 1. The photodetectorreadings for the reference sensor are plotted as line 20 and thephotodetector readings for the sensor LED are plotted as line 22. Alarge increase in the red LED signal 22 is seen in the graph (it changesfrom about 0.6 volt to almost 2 volts) as the carbon dioxide level inthe bottle is increased from 0% CO₂ to 100% CO₂. At the same time, theReference LED signal 20 changes from 2.32 volts to 2.29 volts (a changeof 30 mV), so it is very stable over the course of the LES changingcolor.

In order to study the changes in the optical signal as a function of thebottle position in relation to the optical system, a calibration/testfixture was constructed consisting of a digital micrometer that isattached to the BacT/ALERT bottle. The bottle is first placed in thenormal (home) position in the BacT/ALERT rack assembly so that it is asclose to the optical system as is possible. Readings of the reflectanceare taken, then the bottle is displaced by adjusting the micrometer. Themicrometer provides precise small adjustments to the z-axis displacement(i.e. it moves the bottle further from the optical system) so that theeffects of displacement can be quantified. The normalized change inoptical signal as a function of the displacement is shown graphically inFIG. 5, again with photodetector signal for illumination of thereference LED plotted as line 20 and the photodetector signal for thesensor LED plotted as line 22. It is seen that the displacement causes alinear shift in the signals received by the photodetector. While thesensor LED signal 22 and the reference LED signal 20 have differentslopes of change, each is linear, so that a relationship can bedeveloped to compensate for changes in the signal LED as a function ofchanges in the reference LED detector output, e.g., due to displacementof the bottle from a home or nominal position. Equations were computedfor the graphs in FIG. 5; the equations are listed below in table 2along with the goodness of fit parameter (R2).

TABLE 2 Detector_output (Signal) = 0.2652 − 0.2554x R2 = 0.9963Detector_output(Reference) = 0.5621 − 0.2384x R2 = 0.9999 Where x = thelinear displacement distance (in inches)

Accordingly, by mapping the change in intensity of the reference LED'soutput, a displacement value can be determined. Applying that value tothe signal LED's output, the amount of intensity reduction can bequantified and compensated for.

A further test of the capabilities of the detector arrangement of FIG. 3was performed by injecting a inoculum of Saccharomyces cerevisiae intothe blood culture bottle and monitoring the colorimetric sensor usingthe sensor LED and reference LED optics while the yeast grows in thebottle. FIG. 6 shows the growth curve of the yeast growth—lag,exponential and stationary growth phases are shown. During the growth(and changes in the response of the LES sensor), it is seen that thereference LED signal 20 is unchanging, whereas the sensor LED signal 22changes due to change in CO₂ concentration as a result of microbialgrowth. The flatness of the curve 20 verifies the insensitivity of thephotodetector readings during illumination of the reference LED tochanges in the LES color. It further verifies its ability to monitorchanges in the optical system while not being affected by bacterialgrowth.

FIG. 7 is a block diagram of the electronics 30 for the embodiment ofFIG. 3. The electronics 30 includes an “optical nest” 32 consisting ofthe sensor LED 4, the reference LED 10, and the photodetector 5. Theoutput of the photodetector is converted into a digital signal in an A/Dconverter 34 and fed to a data acquisition system 36. The dataacquisition system sends signals to an LED control board 42 whichincludes control circuits and LED drivers which send signals over theconductors 44 and 46 to cause the LEDs 4 and 10 to illuminate in a timedivision multiplexed manner. Photodetector signals from the dataacquisition system are sent to a computer 38, which may be part of theinstrument incorporating the optical nest 32 of FIGS. 3 and 7.(Incidental electronics such as filters and current-to-voltage converterare omitted in the Figure but may be present in the electronics).

Memory 40 stores the calibration constants and relationships between thereference and signal LED outputs, derived from curves such as FIG. 5 andexplained above in Table 2. For example, the memory 40 stores acalibration relationship between intensity signals for the sensor LED asa function of distance of the bottle from the home position (plot 22 inFIG. 5); the computer 38 compensates for a drop in intensity signalsfrom the sensor LED due to the bottle being positioned a distance awayfrom the home position in accordance with calibration relationships forthe sensor LED and the reference LED.

FIG. 8 is a graph of the duty cycle of the reference LED 10 and sensorLED 4 of FIG. 3, showing the time division multiplexing method ofoperation. The sensor LED on and off states are shown on line 50; thereference LED on and off states are shown in line 42. The width of thepulses representing the duty cycle is not to scale and can vary. In onepossible embodiment the duty factor is 33 percent: ⅓ of the time thereference LED is illuminated, ⅓ of the time the sensor LED isilluminated, and ⅓ of the time neither LED is illuminated to enable a“dark” measurement to be made.

Compensation for dust, drift, changes in the optical system, and agingof the optical materials in the beam path are also possible with thearrangement of FIG. 3. Since these occur over an extended time (expectedto be in the duration of months), they would be very slow changing.Compensation is achieved by saving data points from the initialcalibration (e.g., derived from FIG. 5) and compare the photodetectorsignals for the IR LED 10 emission levels to initial values tocompensate for degradation mechanisms in the optical system. This changewould also be applied to the sensor LED 4. For shorter time period driftevents, changes are monitored in the IR LED 10 which should be verysteady over the growth cycle of bacteria; any changes in the IR LEDperformance cause adjustments in the sensor LED photodetector readingsaccordingly, e.g., using stored calibration relationships.

The appended claims are further statements of the disclosed inventions.

1. A detection arrangement for blood culture bottle incorporating acolorimetric sensor subject to change of color due to change in pH orCO₂ of a sample medium within the blood culture bottle, comprising: asensor LED illuminating the colorimetric sensor; a reference LEDilluminating the colorimetric sensor; a control circuit for selectivelyand alternately activating the sensor LED and the reference LED; and aphotodetector, the photodetector measuring reflectance from thecolorimetric sensor during the selective and alternating illumination ofthe colorimetric sensor with the sensor LED and the reference LED andgenerating intensity signals; wherein the reference LED is selected tohave a peak wavelength of illumination such that the intensity signalsof the photodetector from illumination by the reference LED are notsubstantially affected by changes in the color of the colorimetricsensor.
 2. The detection arrangement of claim 1 wherein the referenceLED has a peak wavelength of illumination of between 750 and 900 nm. 3.The detection arrangement of claim 1, further comprising a computerreceiving the intensity signals, the computer including a memory storinga calibration relationship between intensity signals for the referenceLED as a function of distance of the bottle from a home position inrelation to the detection arrangement.
 4. The detection arrangement ofclaim 4, wherein the memory further stores a calibration relationshipbetween intensity signals for the sensor LED as a function of distanceof the bottle from the home position and wherein the computercompensates a drop in intensity signals from the sensor LED due to thebottle being positioned a distance away from the home position inaccordance with calibration relationships for the sensor LED and thereference LED.
 5. A method for detection of colorimetric sensorincorporated in a blood culture bottle, the colorimetric sensor subjectto change of color due to change in pH or CO₂ of a sample medium withinthe blood culture bottle comprising the steps of: alternately andrepeatedly illuminating the colorimetric sensor with a sensor LED and areference LED; measuring reflectance from the colorimetric sensor due tothe illumination of the colorimetric sensor by the sensor LED andreference LED with a photodetector, the photodetector responsivelygenerating intensity signals; wherein the reference LED is selected tohave a peak wavelength of illumination such that the intensity signalsof the photodetector from illumination by the reference LED are notsubstantially affected by changes in the color of the colorimetricsensor.
 6. The method of claim 5, wherein the reference LED has a peakwavelength of illumination of between 750 and 900 nm.
 7. The method ofclaim 5, further comprising the step of storing in computer memory acalibration relationship between intensity signals for the reference LEDas a function of distance of the bottle from a home position in relationto the sensor LED, reference LED and photodetector.
 8. The method ofclaim 6, further comprising the step of storing in computer memory acalibration relationship between intensity signals for the sensor LED asa function of distance of the bottle from a home position in relation tothe sensor LED, reference LED and photodetector.
 9. The method of claim8, further comprising the step of compensating for a drop in intensitysignals from the sensor LED due to the bottle being positioned adistance away from the home position in accordance with calibrationrelationships for the sensor LED and the reference LED.
 10. The methodof claim 9, wherein the step of compensating comprises the step ofdetermining a displacement value for the bottle using the calibrationrelationship for the reference LED and using the calibrationrelationship for the sensor LED to adjust the intensity signal from thephotodetector to correct for the displacement of the bottle by thedisplacement value.